Antibodies that bind IL-13 mutants

ABSTRACT

This invention provides antibodies that bind mutant human interleukin 13 molecules showing varying specificity for the restricted (IL4 independent) IL13 receptor. The mutant hIL13 molecules include those made by substituting the amino acid residues that occur in the alpha-helix regions of native hIL13 with various other amino acid residues. Some of the mutants retain the ability to bind and cause signaling through IL13 receptors, while other mutants do not.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. patentapplication Ser. number 09/679,710 filed on Oct. 5, 2000, which is acontinuation-in-part of U.S. patent application Ser. number 09/054,711filed on Apr. 3, 1998, now U.S. Pat. No. 6,296,843, and is related toand claims the benefit of U.S. Provisional patent application number60/157,934 filed on Oct. 6, 1999.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This invention was made in part with Government support undergrant CA741145 awarded by the National Institutes of Health. TheGovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Human interleukin 13 (hIL13) is a 114 amino acid cytokinesecreted by activated T cells. Minty et al. (1993) Nature, 362:248-250;and McKenzie et al. (1993) Proc. Natl. Acad. Sci. USA, 90:3735-3739.hIL13 is involved in regulating several different physiologicalresponses. Among these, hIL13 has been shown to downregulate theproduction of cytokines involved in inflammation. Minty et al., supra;and de Waal Malefyt et al. (1993) J. Immunol., 151:6370-6381. It hasalso been shown to upregulate expression of major histo-compatibilityclass II molecules and CD23 on monocytes, and to regulate variousaspects of B cell function De Waal Malefyt et al. (1993) Res. Immunol.144:629-633; McKenzie et al., supra; and de Waal Malefyt et al. (1993)J. Immunol., 151:6370-6381. In addition to regulating cells of theimmune system, IL-13 has also been shown to act on other cell types. Forexample, IL13 has been shown to modulate expression of vascular celladhesion mole-cule-1 (VCAM-1) on endothelial cells. Sironi et al. (1994)Blood, 84:1913-1921; Bochner et al. (1995) J. Immunol., 154:799-803; andSchnyder et al. (1996) Blood, 87:4286-4295.

[0004] Based on its predicted secondary structure, hIL13 has been addedto a growing family of growth hormone-like cytokines that all exhibitbundled alpha-helical core topology. Bamborough et al. (1994) Prot.Engin., 7:1077-1082. Structural analyses indicated that hIL13 is aglobular protein comprised mainly of four alpha-helical regions (helicesA, B, C, and D) arranged in a “bundled core.” Miyajima et al. (1992)Ann. Rev. Immunol., 10, 295-331.

[0005] While dissimilar at the primary amino acid level, hIL13 and humaninterleukin 4 (hIL4) bind and signal through a shared receptor complex.Zurawski et al. (1993) EMBO J., 12:2663-2670; and Tony et al. (1994)Eur. J. Biochem., 225:659-66. This shared receptor is a heterodimer thatincludes a first subunit of approximately 140 kDa termed p410, and asecond subunit of approximately 52 kDa termed α′ or IL13Rα1. Idzerda etal. (1990) J. Exp. Med., 173:861-873; Obiri et al. (1995) J. Biol.Chem., 270:8797-8804; Hilton et al. (1996) Proc. Natl. Acad. Sci. USA,93:497-501; and Miloux et al. (1997) FEBS Letters, 401:163-166. UnlikehIL4, hIL13 does not bind p140 in the absence of α′. Vita et al. (1995)J. Biol. Chem., 270:3512-3517. In addition to the shared receptor,another hIL13 receptor termed the restricted (IL4 independent) receptorexists. In contrast to the shared receptor, the latter receptor bindshIL13 but not hIL4. The restricted receptor is also sometimes called theglioma-associated receptor because it is preferentially expressed athigh levels in certain malignant cells, including high grade humangliomas. Debinski et al. (1995) Clin. Cancer Res., 1:1253-1258; andDebinski et al. (1996) J. Biol. Chem., 271, 22428-22433. In addition tobeing associated with malignancies, hIL13 has also been associated withother pathological conditions. Notably, IL13 has been shown to beinvolved in pathways that regulate airway inflammation, suggesting thatthis cytokine might play an important role in asthma and perhaps otherallergic pathologies. Webb et al., (2000) J. Immunol.165:108-113; andDjukanovic, R. (2000) Clin. Exp. Allergy 30 Suppl 1:46-50.

SUMMARY OF THE INVENTION

[0006] The invention relates to the development and characterization ofseveral mutants of hIL13. Using these mutants, three regions of nativehIL13 were identified as being required for signaling through the sharedreceptor. These regions were localized to alpha-helices A, C and D andwere generally separated from the regions involved in binding to therestricted receptor. Glutamic acids at positions 13 and 16 in hIL13alpha-helix A, arginine and serine at positions 66 and 69 in helix C,and arginine at position 109 in helix D were found to be important ininducing biological signaling because these mutations resulted in theloss and/or gain of functional phenomena.

[0007] Mutants within the invention include those having one or more ofthe native amino acids of hIL13 at positions 13, 16, 17, 66, 69, 99,102, 104, 105, 106, 107, 108, 109, 112, 113, and 114 replaced with adifferent amino acid. These mutants are expressed herein as hIL13X₁PX₂,where P is a number corresponding to the position of the mutated aminoacid in hIL13, X₁ is the letter abbreviation of the amino acid that wasreplaced, and X₂ is the letter abbreviation of the replacement aminoacid. For example, hIL13.E13K represents a mutant form of hIL13 that hasthe glutamic acid residue that naturally occurs at position 13 in nativehIL13 replaced with a lysine residue. Representative mutants within theinvention include hIL13.E13K, hIL13.E13I, hIL13.E13C, hIL13.E13S,hIL13.E13R, hIL13.E13Y, hIL13.E13D, hIL13.E16K, hIL13.E17k, hIL13.R66D,hIL13.S69D, hIL13.D99K, hIL13.L102A, hIL13.L104A, hIL13.K105D,hIL13.K106D, hIL13.L107A, hIL13.F108Y, hIL13.R109D, hIL13.R112D,hIL13.F113D, and hIL13.N114D.

[0008] Also within the invention are compositions including a mutanthIL13 having an amino acid sequence having at least 90% sequenceidentity to native hIL13 (SEQ ID NO:1). Such mutants can have a mutationin a domain corresponding to the A, C, or D alpha-helices of nativehIL13. Exemplary mutants include those with a polypeptide having anamino acid sequence of one of SEQ ID NOs: 2-23.

[0009] Mutants of hIL13 within the invention can be those thatspecifically bind the shared IL4/IL13 receptor but not the restricted(IL4-independent) receptor; those that specifically bind the restricted(IL4-independent) receptor but not the shared IL4/IL13 receptor; orthose that bind both receptors.

[0010] Some hIL13 mutants of the invention specifically bind to an hIL13receptor associated with a cell in a manner that induces a measurablechange in the cell's physiology. This change can be of greater or lessmagnitude than a change in the cell's physiology that would be inducedby specifically binding the IL13 receptor with native hIL13.

[0011] Compositions within the invention can include both an hIL13mutant and a pharmaceutically acceptable carrier.

[0012] Mutants of hIL13 within the invention can be conjugated to aneffector molecule such as a cytotoxin (e.g., Pseudomonas exotoxin,PE38QQR, PE1E, PE4E, Diptheria toxin, ricin, abrin, saporin, andpokeweed viral protein), a detectable label (e.g., radionuclide), anantibody, a liposome, or a lipid.

[0013] In another aspect the invention includes a purified nucleic acidencoding a mutant hIL13. Also within the invention is an antibody thatspecifically binds a mutant hIL13 molecule, but not a native hIL13molecule. And in another aspect, the invention features a method ofdelivering a mutant hIL13 to a cell. The method can include the stepsof: providing a mutant hIL13 and a cell; and contacting the cell withthe mutant hIL13.

[0014] Unless otherwise defined, all technical terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Commonly understood definitions ofmolecular biology terms can be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

[0015] As used herein, the phrase “native hIL13” means the mature formof human interleukin 13, the amino acid sequence of which is shownherein as SEQ ID NO:1.

[0016] The phrase “hIL13 mutant” or a “mutant hIL13 molecule” means anhIL13 in which one or more of the amino acids differ from thecorresponding amino acids in the native hIL13. Thus, for example, wherea native hIL13 has a glutamic acid at position 13, a mutant hIL13 canhave an amino acid other than glutamic acid at position 13 (e.g.,glutamic acid is substituted with lysine). It will appreciated thatmutant IL13 molecules of this invention include mutant IL 13 moleculesof other mammalian species (e.g., rat, murine, porcine, ovine, goats,non-human primates, bovine, canus, and the like) and this inventioncontemplates the use of mutant IL13 in veterinary as well as humanmedical conditions.

[0017] As used herein, the terms “protein” and “polypeptide” are usedsynonymously to mean any peptide-linked chain of amino acids, regardlessof length or post-translational modification, e.g., glycosylation orphosphorylation. An “purified” polypeptide is one that has beensubstantially separated or isolated away from other polypeptides in acell, organism, or mixture in which the polypeptide occurs (e.g., 30,40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).

[0018] As used herein, a “nucleic acid” or a “nucleic acid molecule”means a chain of two or more nucleotides such as RNA (ribonucleic acid)and DNA (deoxyribonucleic acid). A “purified” nucleic acid molecule isone that has been substantially separated or isolated away from othernucleic acid sequences in a cell or organism in which the nucleic acidnaturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99,100% free of contaminants). The term includes, e.g., a recombinantnucleic acid molecule incorporated into a vector, a plasmid, a virus, ora genome of a prokaryote or eukaryote. Examples of purified nucleicacids include cDNAs, fragments of genomic nucleic acids, nucleic acidsproduced polymerase chain reaction (PCR), nucleic acids formed byrestriction enzyme treatment of genomic nucleic acids, recombinantnucleic acids, and chemically synthesized nucleic acid molecules. A“recombinant” nucleic acid molecule is one made by an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

[0019] As used herein, “sequence identity” means the percentage ofidentical subunits at corresponding positions in two sequences when thetwo sequences are aligned to maximize subunit matching, i.e., takinginto account gaps and insertions. When a subunit position in both of thetwo sequences is occupied by the same monomeric subunit, e.g., if agiven position is occupied by an alanine in each of two polypeptidemolecules, then the molecules are identical at that position. Forexample, if 7 positions in a sequence 10 amino acids in length areidentical to the corresponding positions in a second 10 amino acidsequence, then the two sequences have 70% sequence identity. Sequenceidentity is typically measured using sequence analysis software (e.g.,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705).

[0020] By the term “antibody” is meant an immunoglobulin as well as anyportion or fragment of an immunoglobulin whether made by enzymaticdigestion of intact immunoglobulin or by techniques in molecularbiology. The term also refers to a mixture containing an immunoglobulin(or portion or fragment thereof) such as an antiserum.

[0021] The term “specifically binds”, as used herein, when referring toa polypeptide (including antibodies) or receptor, refers to a bindingreaction which is determinative of the presence of the protein orpolypeptide or receptor in a heterogeneous population of proteins andother biologics. Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody), the specified ligand or antibodybinds to its particular “target” (e.g. an IL13 specifically binds to anIL13 receptor) and does not bind in a significant amount to otherproteins present in the sample or to other proteins to which the ligandor antibody may come in contact in an organism. Generally, a firstmolecule that “specifically binds” a second molecule has a bindingaffinity greater than about 10⁵ (e.g., 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,and 10¹² or more) moles/liter for that second molecule.

[0022] A “mutation” in a polypeptide refers to the substitution of anamino acid at a particular position in a polypeptide with a differentamino acid at that position. Thus, for example, the mutation hIL13.E13Kindicates that the native amino acid at position 13 in IL13 (glutamicacid, E) is replaced with lysine (K). In some cases, a mutation can bethe deletion, addition, or substitution of more than one amino acid in apolypeptide. The mutation does not require an actual removal andsubstitution of the amino acid(s) in question. The protein can becreated de novo with the replacement amino acid in the position(s) ofthe desired mutation(s) so the net result is equivalent to thereplacement of the amino acid in question.

[0023] Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol. In addition, the particular embodiments discussed below areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The invention is pointed out with particularity in the appendedclaims. The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

[0025]FIG. 1 is a photograph of a SDS-PAGE (A) and Western blot (B)analysis of purified hIL13 and various hIL13 mutants. Five hundrednanograms of each purified cytokine was loaded per sample. Proteins weredetected using a Coomassie Blue stain, panel A. Separated proteins froma duplicate gel were electroblotted to a PVDF membrane and detected withan anti-hIL13 antibody in a Western blot protocol using an enhancedchemiluminescence detection system, panel B.

[0026]FIG. 2 is circular dichroism (CD) spectra obtained from purifiedhIL13 and various hIL13 mutants. Each protein was diluted in PBS (0.1mg/ml), thermally equilibrated to 37° C., and its CD spectrum recordedover the wavelength range of 185 nm to 260 nm. The CD spectrum ofunfolded hIL13 (panel D) was obtained by diluting the protein in 8 Murea containing 40 mM dithiothreitol prior to analysis. The reportedspectra were the average of three consecutive measurements. The mutantsin each panel, listed from top to bottom, represent the order of thespectra in each panel, from top to bottom.

[0027]FIG. 3 is graphical representations of data obtained fromproliferation assays using TF-1 cells induced with hIL13 and varioushIL13 mutants. TF-1 cells were cultured in the presence of increasingconcentrations of the indicated protein for 72 h. The amount of TF-1cell proliferation, compared to control experiments induced with bufferalone, was determined calorimetrically. The reported data is the averageof triplicate samples with the error bars representing the standarddeviation within a data set. Experiments were repeated several times.Panels represents hIL13 alpha-helix A mutants that increased TF-1 cellproliferation (A), hIL13 alpha-helix A mutants that failed to increaseTF-1 cell proliferation (B), and hIL13 alpha-helix C mutants that failedto increase TF-1 cell proliferation (C).

[0028]FIG. 4 is a series of photomicrographs of indirectimmunofluorescence analyses of HUVEC for VCAM-1 expression induced byhIL13 and various hIL13 mutants. Panels A-F and G-J are from twoseparate experiments, each with its own set of controls. HUVEC cellswere cultured overnight in media containing buffer alone (panels A andG) or 1 mg/ml of either wild-type hIL13 (panels B and H) or variousmutants (panels C-F, I and J). Induced expression of the protein wasdetected through a rhodamine filter using goat anti-VCAM-1 IgG primaryantibody and rabbit anti-goat IgG CY3-conjugated secondary antibody. Thesensitivity of the imaging camera was set to detect the level offluorescence in the control field, panels A and G. No furtheradjustments were made to the sensitivity, allowing for the amount ofincreased or decreased fluorescence in the experimental fields to bedirectly related to the amount of interleukin-induced VCAM-1 expression.Photomicrographs are shown at 20×magnification (20×).

[0029]FIG. 5 is graphical representations of data obtained fromcytotoxicity assays performed to assess the ability of hIL13 and hIL13mutants to block the killing of U-251MG (panel A) and SNB-19 (panel B)cells by hIL13-PE1E. Cultured cells were incubated with buffer alone,shown in all panels by closed diamonds, or 1 mg/ml of hIL13 or theindicated mutant for 1 hour at 37° C., prior to the addition ofincreasing concentrations of hIL13-PE1E. The reported data is theaverage of triplicate samples with the error bars representing thestandard deviation within a data set. Experiments were repeated severaltimes.

DETAILED DESCRIPTION

[0030] This invention encompasses compositions and methods relating tohIL13 mutants. The below described preferred embodiments illustrateadaptations of these compositions and methods. Nonetheless, from thedescription of these embodiments, other aspects of the invention can bemade and/or practiced based on the description provided below.

[0031] Biological Methods

[0032] Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates). Varioustechniques using polymerase chain reaction (PCR) are described, e.g., inInnis et al., PCR Protocols: A Guide to Methods and Applications,Academic Press: San Diego, 1990. PCR-primer pairs can be derived fromknown sequences by known techniques such as using computer programsintended for that purpose (e.g., Primer, Version 0.5, ©1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). The ReverseTranscriptase Polymerase Chain Reaction (RT-PCR) method used to identifyand amplify certain polynuleotide sequences within the invention wasperformed as described in Elek et al., In Vivo, 14:172-182, 2000).Methods for chemical synthesis of nucleic acids are discussed, forexample, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981,and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemicalsynthesis of nucleic acids can be performed, for example, on commercialautomated oligonucleotide synthesizers. Immunological methods (e.g.,preparation of antigen-specific antibodies, immunoprecipitation, andimmunoblotting) are described, e.g., in Current Protocols in Immunology,ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods ofImmunological Analysis, ed. Masseyeff et al., John Wiley & Sons, NewYork, 1992.

[0033] Mutant hIL13 Molecules

[0034] The mutant hIL13 molecules of the invention are based on theamino acid sequence of native hIL13 (SEQ ID NO:1). The hIL13 mutantswithin the invention differ by one or more amino acids from nativehIL13. For example, hIL13 mutants within the invention can have 90% ormore (e.g., 91, 92, 93, 94, 95, 96, 97, 98, and 99%) sequence identitywith native hIL13. Examples of hIL13 mutants within the invention arethose having the amino acid sequences of SEQ ID NOs:2-23. These mutantseach have a mutation in a domain corresponding to either the A (residues9-25 of SEQ ID NO:1), C (residues 59-71 of SEQ ID NO:1), or D (residues97-113 of SEQ ID NO:1) alpha-helices of native hIL13. Each of thesefeatures a substitution of one of the amino acid residues that occurs innative hIL13. Other hIL13 mutants within the invention are those withtwo or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) such amino acidsubstitutions, as well as deletion (e.g., truncation) and addition(i.e., those with additional amino acids added to the native hIL13sequence) mutations.

[0035] Mutants of hIL13 can be made in a number of ways by adaptingtechniques well known in the art. See, e.g., Sambrook et al., supra; andAusubel et al., supra. For example, starting with the known amino acidsequence of hIL13 (i.e., SEQ ID NO:1), the skilled artisan canchemically synthesize various mutant hIL13 molecules using, e.g,automated commercial polypeptide synthesizers. Techniques for solidphase synthesis of polypeptides are well known. See, e.g., Barany andMerrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156(1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed.Pierce Chem. Co., Rockford, Ill. (1984). Using this technique, hIL13mutants can be synthesized as a single polypeptide. Alternatively,shorter oligopeptide portions of the mutant hIL13 molecule can first besynthesized and then fused together to form the full length mutant bycondensation of the amino terminus of one oligopeptide portion with thecarboxyl terminus of the another oligopeptide portion to forming apeptide bond. The fusions can then be purified by standard proteinchemistry techniques.

[0036] Mutants of hIL13 can also be produced through recombinantexpression of hIL13-encoding nucleic acids (see below) in which thenucleic acid is modified, randomly or in a site-specific manner, tochange (substitute), add to, or delete, some or all of the amino acidsin the encoded polypeptide. Site-specific mutations can be introducedinto the IL13-encoding nucleic acid by a variety of conventionaltechniques well described in the scientific and patent literature.Illustrative examples include: site-directed mutagenesis by overlapextension polymerase chain reaction (OE-PCR), as in Urban (1997) NucleicAcids Res. 25: 2227-2228; Ke (1997) Nucleic Acids Res., 25: 3371-3372,and Chattopadhyay (1997) Biotechniques 22: 1054-1056, describingPCR-based site-directed mutagenesis “megaprimer” method; Bohnsack (1997)Mol. Biotechnol. 7: 181-188; Ailenberg (1997) Biotechniques 22: 624-626,describing site-directed mutagenesis using a PCR-based staggeredre-annealing method without restriction enzymes; Nicolas (1997)Biotechniques 22: 430-434, site-directed mutagenesis using longprimer-unique site elimination and exonuclease III. Unique-siteelimination mutagenesis can also be used (see, e.g., Dang et al. (1992)Anal. Biochem., 200: 81). The production of mutants of biologicallyactive proteins such as IFN-beta and IL-2 is described in detail in U.S.Pat. No. 4,853,332 and the mutation of hIL13 is described in Example 1below.

[0037] Other hIL13 mutants can be prepared by chemically modifyingnative hIL13 according to known chemical modification methods. See,e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995)Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33:7886-7896. Likewise, hIL13 mutants made by chemical synthesis or byexpression of nucleic acids as described above can be chemicallymodified to make additional hIL13 mutants.

[0038] Characterizing hIL13 Mutants

[0039] Mutants of hIL13 can have characteristics that differ from thosenative hIL13. For example, native hIL13 has the functionalcharacteristics of binding both shared receptor and the restrictivereceptor. Native hIL13 also has the characteristic of inducingtransmembrane signals through binding shared receptors expressed on acell surface. Such signaling can result in a measurable change in thecell's physiology. Changes can be the production of secondmessengers—e.g, an increase in intracellular [Ca²⁺], activation ofprotein kinases and/or phosphorylases, changes in phosphorylation of asubstrate, changes in signal transducers and activators oftranscription, etc. They can also be changes in the cell proteome, e.g.,from increased or decreased transcription or translation. Or they can bechanges in a functional or phenotypic characteristic of the cell. Forinstance, adding native hIL13 to TF-1 cells can increase their rate ofproliferation. As another example, adding native hIL13 can cause HUVECto increase their expression of VCAM-1.

[0040] Characteristics of a given mutant hIL13 molecule can therefore beassessed by examining the ability of the molecule to bind the sharedreceptor and/or the restrictive receptor. Similarly, the ability of themutant molecule to induce transmembrane signaling can be assessed byexamining whether contacting a cell expressing an IL13 receptor with themutant molecule results in a change in the cell's physiology. By thesemethods, hIL13 mutants can be characterized as those that bind both theshared receptor and/or the restrictive receptor, those that bind onlyone of the receptors, and those that do not bind either receptor. Byquantifying the affinity of a mutant hIL13 molecule, it can also becharacterized as one that binds with less, about equal, or more affinitythan native hIL13. Mutants of hIL13 can also be characterized as havingor lacking the ability to cause a transmembrane signal and/or a changein a cell's function or phenotype. The changes caused by a mutant hIL3molecule can also be quantified to further characterize the molecule asone that causes such changes less than (of less magnitude), about equalto, or more than (of greater magnitude) those caused by native hIL13.For instance mutants of hIL13 that specifically bind to an hIL13receptor associated with a cell in a manner that induces a measurablechange in the cell's physiology can be those that modulate theproliferation rate of a cell line that expresses an IL13 receptor suchas TF-1 cells. Antagonistic hIL13 mutants are those that reduce theproliferation rate of the cell line compared to that induced by nativehIL13; agonistic hIL13 mutants are those that induce about same (e.g.,50-150% or 75-125% of) proliferation rate of the cell line as thatinduced by native hIL13; and superagonistic hIL13 mutants are those thatincrease the proliferation rate of the cell line compared to thatinduced by native hIL13. See Examples, below.

[0041] Chimeric Molecules of Mutant hIL13 and Effector Molecules

[0042] The invention also provides a chimeric molecule including amutant hIL13 molecule conjugated to an effector molecule. The effectormolecule can be any molecule that can be conjugated to an hIL13 mutantand exert a particular function. Examples of effector molecules includecytotoxins, drugs, detectable labels, targeting ligands, and deliveryvehicles.

[0043] A mutant hIL13 molecule conjugated with a one or more cytoxinscan be used to kill cells expressing a receptor to which the mutantbinds. Cytotoxins for use in the invention can be any cytotoxic agent(i.e., molecule that can kill a cell after contacting the cell) that canbe conjugated to hIL13 or an hIL13 mutant. Examples of cytotoxinsinclude, without limitation, radionuclides (e.g., ³⁵S, ¹⁴C, ³²P, ¹²⁵I,¹³¹I, ⁹⁰Y, ⁸⁹Zr, ²⁰¹Tl, ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁷Cu, ²¹³Bi, ²¹¹At, etc,conjugated radionuclides, and chemotherapeutic agents. Further examplesof cytotoxins include, but are not limited to, antimetabolites (e.g.,5-flourouricil (5-FU), methotrexate (MTX), fludarabine, etc.),anti-microtubule agents (e.g., vincristine, vinblastine, colchicine,taxanes (such as paclitaxel and docetaxel), etc.), alkylating agents(e.g., cyclophasphamide, melphalan, bischloroethylnitrosurea (BCNU),etc.), platinum agents (e.g., cisplatin (also termed cDDP), carboplatin,oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g., doxorubicin,daunorubicin, etc.), antibiotic agents (e.g., mitomycin-C),topoisomerase inhibitors (e.g., etoposide, tenoposide, andcamptothecins), or other cytotoxic agents such as ricin, diptheria toxin(DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin, pokeweed viralprotein, ethidium bromide, glucocorticoid, and others. See, e.g. U.S.Pat. No. 5,932,188. Useful variations of PE and DT include PE38QQR (see,U.S. Pat. No. 5,614,191), PE1E and PE4E (see, e.g., Chaudhary et al(1995) J. Biol. Chem., 265:16306), and DT388 and DT398 (Chaudhary, etal. (1991) Bioch. Biophys. Res. Comm., 180: 545-551) can also be used.

[0044] Mutant hIL13 molecules conjugated with one or more detectablelabels can be used to detect the presence of a receptor to which themutant binds, e.g., in diagnostic assays (e.g., in the detection of shedtumor cells overexpression the IL13 receptor) and/or in the in vivolocalization of tumor cells. Detectable labels for use in the inventioncan be any substance that can be conjugated to hIL13 or an hIL13 mutantand detected. Suitable detectable labels are those that can be detected,for example, by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful detectablelabels in the present invention include biotin or streptavidin, magneticbeads (e.g., Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga,⁶⁸Ga, or ⁷²As,), radioopaque substances such as metals for radioimaging,paramagnetic agents for magnetic resonance imaging, enzymes (e.g.,horseradish peroxidase, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

[0045] Means of detecting such labels are well known to those of skillin the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photo detector to detect emitted illumination.Enzymatic labels are typically detected by providing the enzyme with asubstrate and detecting the reaction product produced by the action ofthe enzyme on the substrate, and colorimetric labels are detected bysimply visualizing the colored label, and so forth.

[0046] Mutant hIL13 molecules conjugated with one or more targetingligands (i.e., molecules that can bind a particular receptor) can beused to mediate binding of the mutants to a particular receptor or cellexpressing the receptor. Any targeting ligand that can be conjugated tohIL13 or an hIL13 mutant can be used. Examples of such targeting ligandsincludes antibodies (or the antigen-binding portion of antibodies); andchemokines, growth factors, soluble cytokine receptors (e.g., thoselacking a transmembrane domain), superantigens, or other molecules thatbind a particular receptor. A large number of these molecules are known,e.g., IL-2, IL-4, IL-6, IL-7, tumor necrosis factor (TNF), anti-Tac,TGF-alpha., SEA, SEB, and the like. As a representative example, anhIL13 mutant can be conjugated with a soluble form of a hIL13 receptor.This conjugate, for example, could be used to both antagonize anendogenous hIL13 receptor on a cell and neutralize any hIL13 present inthe vicinity of the cell.

[0047] Mutant hIL13 molecules conjugated with one or more nucleic acidscan be used to specifically target delivery of the nucleic acid(s) to atarget cell (e.g., one expressing an receptor to which the mutantbinds). Any nucleic acid that can be conjugated to hIL13 or an hIL13mutant can be used. The nucleic acids can be attached directly to themutant hIL13, attached via a linker, or complexed with or encapsulatedin another moiety (e.g., a lipid, a liposome, a viral coat, or the like)that is attached to the mutant IL13 molecule. The nucleic acid canprovide any of number of effector functions. For example, a nucleic acidencoding one or more proteins can be used to deliver a particularenzymatic activity, substrate, and/or epitope to a target cell. Forthese applications or others where expression (e.g. transcription ortranslation) of the nucleic acid is desired, the nucleic acid ispreferably a component of an expression cassette that includes all theregulatory sequences necessary to express the nucleic acid in the cell.Suitable expression cassettes typically include promoter initiation andtermination codons, and are selected to optimize expression in thetarget cell. Methods of constructing suitable expression cassettes arewell known to those of skill in the art. See, e.g., Sambrook et al.,supra.

[0048] A mutant hIL13 molecule conjugated with a one or more drugs canbe used to deliver such drug(s) to cells expressing a receptor to whichthe mutant binds. Any drug which can be conjugated to hIL13 or an hIL13mutant can be used. Examples of such drugs include sensitizing agentsthat render a target (e.g., tumor) cell susceptible to various cancertherapeutics. The sensitizing agent can be a small molecule drug or agene (under the control of a promoter in an appropriate expressioncassette to induce expression in the target cell). For example, it hasbeen proposed that expression of the herpes simplex virus (HSV)thymidine kinase (TK) gene in proliferating cells, renders the cellssensitive to the deoxynucleoside analog, ganciclovir. Moolten et at.(1986) Cancer Res. 46:5276-5281; Moolten et al. (1990) Hum. Gene Ther.1: 125-134; Moolten et al. (1990) J. Natl. Cancer Inst. 82: 297-300;Short et al. (1990) J. Neurosci. Res. 27:427-433; Ezzedine et al. (1991)New Biol. 3: 608-614, Boviatsis et al. (1994) Hum. Gene Ther. 5:183-191. HSV-TK mediates the phosphorylation of ganciclovir, which isincorporated into DNA strands during DNA replication (S-phase) in thecell cycle, leading to chain termination and cell death. Elion (1983)Antimicr. Chemother. 12, sup. B:9-17. A second example of a gene with adrug-conditional “killing” function is the bacterial cytosine deaminasegene, which confers chemosensitivity to the relatively non-toxic5-fluorouracil precursor 5-fluorocytosine. Mullen et al (1992) Proc.Natl. Acad. Sci. USA 89: 33-37; Huber et al. (1993) Cancer Res. 53:4619-4626; Mullen et al. (1994) Cancer Res. 54: 1503-1506. Still anotherexample of a gene with a drug-conditional “killing” function is acytochrome P450 gene. Expression, of the gene product renders tumorcells sensitive to a chemotherapeutic agent, in particular,cyclophosphamide or ifosphamide. See, U.S. Pat. No. 5,688,773. The drugemployed need not be a gene. For example, it can be one of the compoundsthat can treat multiple drug resistance of susceptible tumor cellsdescribed in U.S. Pat. No. 4,282,233. Other drugs can also be used. Forexample, chemotherapy drugs such as doxorubicin, vinblastine, genistein,and other described above can be conjugated to the mutant hIL13molecule.

[0049] A mutant hIL13 molecule conjugated to a one or more deliveryvehicles is also within the invention. Such conjugates can be used todeliver other substances such as a drug to cells expressing a receptorto which the mutant binds. Any delivery vehicle that can be conjugatedto hIL13 or an hIL13 mutant can be used. Examples of such deliveryvehicles include liposomes and lipids (e.g., micelles). Liposomesencapsulating drugs or micelles including drugs may also be used.Methods for preparing liposomes attached to proteins are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735;and Connor et al., Pharm. Ther., 28: 341-365 (1985).

[0050] Effector molecules can be conjugated (e.g., covalently bonded) toa mutant hIL13 by any method known in the art for conjugating two suchmolecules together. For example, the mutant hIL13 can be chemicallyderivatized with an effector molecule either directly or using a linker(spacer). Several methods and reagents (e.g., cross-linkers) formediating this conjugation are known. See, e.g., catalog of PierceChemical Company; and Means and Feeney, Chemical Modification ofProteins, Holden-Day Inc., San Francisco, Calif. 1971. Variousprocedures and linker molecules for attaching various compoundsincluding radionuclide metal chelates, toxins, and drugs to proteins(e.g., to antibodies) are described, for example, in European PatentApplication No. 188,256; U.S. Pat. Nos. 4,671,958; 4,659,839; 4,414,148;4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al.Cancer Res. 47: 4071-4075 (1987). In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982); Waldmann (1991) Science, 252: 1657; and U.S.Pat. Nos. 4,545,985 and 4,894,443.

[0051] Where the effector molecule is a polypeptide, the chimericmolecule including the hIL13 mutant and the effector can be a fusionprotein. Fusion proteins can be prepared using conventional techniquesin molecular biology to join the two genes in frame into a singlenucleic acid, and then expressing the nucleic acid in an appropriatehost cell under conditions in which the fusion protein is produced.

[0052] A mutant hIL13 may be conjugated to one or more effectormolecule(s) in various orientations. For example, the effector moleculemay be joined to either the amino or carboxy termini of the mutanthIL13. The mutant IL13 molecule may also be joined to an internal regionof the effector molecule, or conversely, the effector molecule may bejoined to an internal location of the mutant IL13 molecule.

[0053] In some circumstances, it is desirable to free the effectormolecule from the mutant hIL13 molecule when the chimeric molecule hasreached its target site. Therefore, chimeric conjugates comprisinglinkages that are cleavable in the vicinity of the target site may beused when the effector is to be released at the target site. Cleaving ofthe linkage to release the effector molecule from the mutant IL13molecule may be prompted by enzymatic activity or conditions to whichthe conjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used. A numberof different cleavable linkers are known to those of skill in the art.See, e.g., U.S. Pat. Nos. 4,618,492; 4,542,225; and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven effector molecule to a mutant hIL13 molecule.

[0054] Nucleic Acids Encoding Mutant hIL13 Molecules and Methods ofMaking Mutant hIL13 Molecules Using Nucleic Acids

[0055] The invention also provides purified nucleic acids encoding themutant hIL13 molecules and the fusion proteins described above. Startingwith a known protein sequence, DNA encoding the mutant hIL13 moleculesor the fusion proteins may be prepared by any suitable method,including, for example, cloning and restriction of appropriate sequencesor direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al. (1979) Meth. Enzymol. 68: 90-99; thephosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151;the diethylphosphoramidite method of Beaucage et al. (1981) Tetra.Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No.4,458,066. Because of the degeneracy of the genetic code, a large numberof different nucleic acids will encode the mutant hIL13 molecules andthe fusion proteins. Each of these is included within the invention.

[0056] Chemical synthesis produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. Longer DNA sequences may be obtained bythe ligation of shorter sequences. Alternatively, subsequences may becloned and the appropriate subsequences cleaved using appropriaterestriction enzymes. The fragments may then be ligated to produce thedesired DNA sequence.

[0057] DNA encoding the mutant hIL13 molecules or the fusion proteinsmay be cloned using DNA amplification methods such as polymerase chainreaction (PCR). Thus, in a preferred embodiment, the gene for hIL13 isPCR amplified, using primers that introduce one or more mutations. Theprimers preferably include restrictions sites, e.g., a sense primercontaining the restriction site for NdeI and an antisense primercontaining the restriction site for HindIII. In one embodiment, theprimers are selected to amplify the nucleic acid starting at position19, as described by McKenzie et al. (1987), supra. This produces anucleic acid encoding the mature IL13 sequence (or mutant hIL13molecules) and having terminal restriction sites.

[0058] For making DNA encoding the fusion proteins, the DNA encoding theeffector molecule can be obtained from available sources. For example,the PE38QQR fragment may be excised from the plasmid pWDMH4-38QQR orplasmid pSGC242FdNl as described by Debinski et al. Int. J. Cancer, 58:744-748 (1994), and by Debinski et al. ( 1994) Clin. Cancer Res.1:1015-1022 respectively. Ligation of the mutant IL13 molecule and aPseudomonas exotoxin (e.g., PE38QQR) sequences and insertion into avector produces a vector encoding the mutant IL13 joined to the terminusof the Pseudomonas exotoxin (e.g., joined to the amino terminus ofPE38QQR, PE1E, or PE4E (position 253)). In a preferred embodiment, thetwo molecules are joined directly. Alternatively there can be anintervening peptide linker (e.g., a three amino acid junction consistingof glutamic acid, alanine, and phenylalanine introduced by therestriction site).

[0059] While the two molecules are preferably essentially directlyjoined together, one of skill will appreciate that the molecules may beseparated by a peptide spacer consisting of one or more amino acids.Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the solubility, folding, net charge, or hydrophobicity.

[0060] The nucleic acid sequences encoding the mutant hIL13 molecules orthe fusion proteins may be expressed in a variety of host cells,including E. coli, other bacterial hosts, yeast, and various highereukaryotic cells such as the COS, CHO and HeLa cells lines and myelomacell lines. The recombinant protein gene will be operably linked toappropriate expression control sequences for each host. F or E. colithis includes a promoter such as the T7, trp, or lambda promoters, aribosome binding site and preferably a transcription termination signal.For eukaryotic cells, the control sequences will include a promoter andpreferably an enhancer derived from immunoglobulin genes, SV 40,cytomegalovirus, etc., and a polyadenylation sequence, and may includesplice donor and acceptor sequences.

[0061] Plasmid vectors of the invention made as described above can betransferred into the chosen host cell by well-known methods such ascalcium chloride, or heat shock, transformation for E. coli and calciumphosphate treatment or electroporation for mammalian cells. Cellstransformed by the plasmids can be selected by resistance to antibioticsconferred by genes contained on the plasmids, such as the amp, gpt, neoand hyg genes.

[0062] Once expressed, the recombinant mutant hIL13 molecules or fusionproteins can be purified according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like. See, generally, R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); andDeutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990). Substantially purecompositions of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity are most preferred for pharmaceuticaluses. Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically.

[0063] After chemical synthesis, biological expression, or purification,the mutant hIL13 molecules or the fusion proteins may possess aconformation substantially different than the native conformations ofthe constituent polypeptides. In this case, it may be necessary todenature and reduce the polypeptide and then to cause the polypeptide tore-fold into the preferred conformation. Methods of reducing anddenaturing proteins and inducing re-folding are well known to those ofskill in the art. See, Debinski et al. (1993) J. Biol. Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585;and Buchner, et al. (1992) Anal. Biochem., 205: 263-270.

[0064] Modifications can be made to the IL13 receptor targeted fusionproteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons.

[0065] Antibodies

[0066] Mutants of hIL13 (or immunogenic fragments or analogs thereof)can be used to raise antibodies useful in the invention. Suchpolypeptides can be produced by recombinant techniques or synthesized asdescribed above. In general, hIL13 mutants can be coupled to a carrierprotein, such as KLH, as described in Ausubel et al., supra, mixed withan adjuvant, and injected into a host mammal. Antibodies produced inthat animal can then be purified by peptide antigen affinitychromatography. In particular, various host animals can be immunized byinjection with an hIL13 mutant or an antigenic fragment thereof.Commonly employed host animals include rabbits, mice, guinea pigs, andrats. Various adjuvants that can be used to increase the immunologicalresponse depend on the host species and include Freund's adjuvant(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Other potentially useful adjuvants include BCG (bacilleCalmette-Guerin) and Corynebacterium parvum.

[0067] Polyclonal antibodies are heterogeneous populations of antibodymolecules that are contained in the sera of the immunized animals.Antibodies within the invention therefore include polyclonal antibodiesand, in addition, monoclonal antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, and molecules produced using a Fabexpression library. Monoclonal antibodies, which are homogeneouspopulations of antibodies to a particular antigen, can be prepared usingthe mutants of hIL13 described above and standard hybridoma technology(see, for example, Kohler et al., Nature 256:495, 1975; Kohler et al.,Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292,1976; Hammerling et al., “Monoclonal Antibodies and T Cell Hybridomas, ”Elsevier, N.Y., 1981; Ausubel et al., supra). In particular, monoclonalantibodies can be obtained by any technique that provides for theproduction of antibody molecules by continuous cell lines in culturesuch as described in Kohler et al., Nature 256:495, 1975, and U.S. Pat.No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al.,Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA80:2026, 1983), and the EBV-hybridoma technique (Cole et al.,“Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96, 1983). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. A hybridomaproducing a mAb of the invention may be cultivated in vitro or in vivo.The ability to produce high titers of mabs in vivo makes this aparticularly useful method of production.

[0068] Once produced, polyclonal or monoclonal antibodies can be testedfor specific recognition of the mutants by Western blot orimmunoprecipitation analysis by standard methods, for example, asdescribed in Ausubel et al., supra. Antibodies that specificallyrecognize and bind to hIL13 mutants are useful in the invention. Forexample, such antibodies can be used to monitor the amount of an hIL13mutant associated with a cell or to block binding of a particular mutanta receptor.

[0069] Antibodies of the invention can be produced using fragments ofthe hIL13 mutants that lie outside highly conserved regions and appearlikely to be antigenic, by criteria such as high frequency of chargedresidues. Cross-reactive anti-hIL13 mutant antibodies are produced usinga fragment of a hIL13 mutant that is conserved amongst members of thisfamily of proteins. In one specific example, such fragments aregenerated by standard techniques of PCR, and are then cloned into thepGEX expression vector (Ausubel et al., supra). Fusion proteins areexpressed in E.coli and purified using a glutathione agarose affinitymatrix as described in Ausubel, et al., supra. Non-cross reactiveantibodies can be prepared by adsorbing the antibody with the antigen(s)that the antibody is desired not to react with. For example, antiseraprepared against a particular hIL13 mutant can be adsorbed with otherhIL13 mutants and/or native hIL13 to reduce or eliminatecross-reactivity.

[0070] In some cases it may be desirable to minimize the potentialproblems of low affinity or specificity of antisera. In suchcircumstances, two or three fusions can be generated for each protein,and each fusion can be injected into at least two rabbits. Antisera canbe raised by injections in a series, preferably including at least threebooster injections. Antiserum is also checked for its ability toimmunoprecipitate recombinant mutants of hIL13 or control proteins, suchas glucocorticoid receptor, CAT, or luciferase.

[0071] Techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against an hIL13 mutant, or afragment thereof. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

[0072] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)₂ fragments that can be produced bypepsin digestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alter-natively, Fab expression libraries can be constructed (Huse etal., Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0073] The antibodies of the invention can be used, for example, in thedetection of a hIL13 mutant in a biological sample. Antibodies can alsobe used to interfere with the interaction of an hIL13 mutant and othermolecules that bind the mutant (e.g., an hIL13 receptor).

[0074] Methods of Delivering a Mutant hIL13 Molecule to a Cell

[0075] The invention also provides a method of delivering an IL13 mutantto a cell. This method is useful, among other things, for directing achimeric molecule including the hIL13 mutant and an effector molecule toa cell so that the effector molecule can exert its function. Forexample, an hIL13 mutant conjugated to a cytotoxin can be delivered to atarget cell to be killed by mixing a composition containing the chimericmolecule with the target cell expressing a receptor that binds themutant. As another example, an hIL13 mutant conjugated to a detectablelabel can be directed to a target cell to be labeled by mixing acomposition containing the chimeric molecule with the target cellexpressing a receptor that binds the mutant.

[0076] Mutant hIL13 molecules can be delivered to a cell by any knownmethod. For example, a composition containing the hIL13 mutant can beadded to cells suspended in medium. Alternatively, a mutant hIL13 can beadministered to an animal (e.g., by a parenteral route) having a cellexpressing a receptor that binds the mutant so that the mutant binds tothe cell in situ. The mutant ILI3 molecules of this invention areparticularly well suited as targeting moieties for binding tumor cellsbecause tumor cells overexpress ILI3 receptors. In particular, carcinomatumor cells (e.g. renal carcinoma cells) overexpress ILI3 receptors atlevels ranging from about 2100 sites/cell to greater than 150,000 sitesper cell. Similarly, gliomas and other transformed cells alsooverexpress ILI3 receptors (ILI3R). Thus, the methods of this inventioncan be used to target an effector molecule to a variety of cancers. Suchcancers are well known to those of skill in the art and include, but arenot limited to, cancers of the skin (e.g., basal or squamous cellcarcinoma, melanoma, Kaposi's sarcoma, etc.), cancers of thereproductive system (e.g., testicular, ovarian, cervical), cancers ofthe gastrointestinal tract (e.g., stomach, small intestine, largeintestine, colorectal, etc.), cancers of the mouth and throat (e.g.esophageal, larynx, oropharynx, nasopharynx, oral, etc.), cancers of thehead and neck, bone cancers, breast cancers, liver cancers, prostatecancers (e.g., prostate carcinoma), thyroid cancers, heart cancers,retinal cancers (e.g., melanoma), kidney cancers, lung cancers (e.g.,mesothelioma), pancreatic cancers, brain cancers (e.g. gliomas,medulloblastomas, meningiomas, etc) and cancers of the lymph system(e.g. lymphoma). In a particularly preferred embodiment, the methods ofthis invention are used to target effector molecules to brain cancers(especially gliomas).

[0077] One of skill in the art will appreciate that identification andconfirmation of ILI3 overexpression by other cells requires only routinescreening using well-known methods. Typically this involves providing alabeled molecule that specifically binds to the ILI3 receptor (e.g., anative or mutant ILI3). The cells in question are then contacted withthis molecule and washed. Quantifying the amount of label remainingassociated with the test cell provides a measure of the amount of ILI3receptor (ILI3R) present on the surface of that cell. In a preferredembodiment, ILI3 receptor may be quantified by measuring the binding of¹²⁵I-labeled IL13 (¹²⁵I-ILI3) to the cell in question. Details of such abinding assay are provided in U.S. Pat. No. 5,614,191.

[0078] As IL13 has been implicated in playing an important regulatoryrole in allergic hyperactivity reactions such as asthma (Webb et al.(2000) J. Immunol. 165:108-113), the invention also provides a method ofmodulating an allergic response by contacting a cell important in theresponse (e.g., a lymphocyte such as a B cell, an eosinophil, a mastcell, and/or any other cells involved in Th₂-dominated inflammatoryresponses) with one or more hIL13 mutants. Thus, for example, whereinteraction of native hIL13 with an hIL13 receptor expressed on a cellcauses transmembrane signals that contribute to the cell's role in anallergic reaction (e.g., inducing inflammation), a mutant hIL13 can beused to block this interaction and inhibit the allergic reaction. Theinteraction between native hIL13 and the IL13 receptor can be blocked,for example, by contacting the cell 1 can with an hIL13 mutant thatbinds to the IL13 receptor (in some cases with more affinity than nativehIL13) but does not cause the transmembrane signaling through thereceptor. For asthma, such an hIL13 mutant could be administered byinhalation of a pharmaceutical composition containing the mutant.

[0079] Pharmaceutical Compositions

[0080] The mutant hIL13 molecules (including those conjugated with aneffector molcule) of this invention can be prepared for parenteral,topical, oral, or local administration, such as by aerosol ortransdermally, for prophylactic and/or therapeutic treatment. Thepharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include powder,tablets, pills, capsules and lozenges. It some cases it may be desirableto protect the fusion proteins and pharmaceutical compositions of thisinvention, from being digested (e.g., when administered orally). Thiscan be accomplished either by complexing the protein with a compositionthat renders it resistant to acidic and enzymatic hydrolysis, or bypackaging the protein in an appropriately resistant carrier such as aliposome. Means of protecting compounds from digestion are well known inthe art (see, e.g., U.S. Pat. No. 5,391,377 describing lipidcompositions for oral delivery of therapeutic agents).

[0081] The pharmaceutical compositions can also be delivered to ananimal by inhalation by any presently known suitable technique. Forexample, the hIL13 mutants of the invention can be a delivered in theform of an aerosol spray produced from pressurized packs or a nebulizer,with the use of a suitable propellant such as dichlorodifluromethane,trichlorotri-fluoromethane, dichlorotetraflurorethane, carbon dioxide,or any other suitable gas. In the case of a pressurized aerosol, thedosage unit may be controlled using a valve to deliver a metered amount.Capsules and cartridges (e.g., of gelatin) containing a powder mix ofthe hIL13 mutant and a suitable base (e.g., lactose or starch) can beused in an inhaler or insufflator to deliver the mutant to therespiratory tract of an animal.

[0082] The pharmaceutical compositions of this invention areparticularly useful for parenteral administration, such as intravenousadministration or administration into a body cavity or lumen of anorgan. The compositions for administration will commonly comprise asolution of the mutant hIL13 molecule dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter (e.g.,pyrogens). These compositions may be sterilized by conventional, wellknown sterilization techniques. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of the mutant hIL13 in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the patient's needs.Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as Remington's PharmaceuticalScience, 15th ed., Mack Publishing Company, Easton, Pa. (1980).

[0083] Toxicity and therapeutic efficacy of the pharmaceuticalcompositions utilized in the invention can be determined by standardpharmaceutical procedures, using either cells in culture or experimentalanimals to determine the LD₅₀ (the dose lethal to 50% of the population)and the ED₅₀ (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Dosesthat exhibit large therapeutic indices are preferred. While those thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets the pharmaceutical composition to the siteof affected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

[0084] The data obtained from cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such pharmaceutical compositions lies preferably within a range ofcirculating concentrations that include an ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anypharmaceutical composition used in a method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve anlC₅₀ (that is, the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography. Although dosage should be determinedfor each particular application, it is expected that a dose of a typicalpharmaceutical composition for intravenous administration would be about0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mgper patient per day may be used, particularly when the pharmaceuticalcompositions is administered to a secluded site and not into the bloodstream, such as into a body cavity or into a lumen of an organ.

[0085] The compositions containing the present hIL13 mutants, or acocktail thereof (i.e., with other proteins), can be administered fortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of the proteins of this invention toeffectively treat the patient.

[0086] Among various uses of the cytotoxic fusion proteins of thepresent invention are included a variety of disease conditions caused byspecific human cells that may be eliminated by the toxic action of theprotein. One preferred application is the treatment of cancer (e.g., aglioma), such as by the use of an mutant IL13 ligand attached to acytotoxin (e.g., PE or a PE derivative).

[0087] It will be appreciated by one of skill in the art that there aresome regions that are not heavily vascularized or that are protected bycells joined by tight junctions and/or active transport mechanisms whichreduce or prevent the entry of macromolecules present in the bloodstream. For example, systemic administration of therapeutics to treatgliomas, or other brain cancers, is constrained by the blood-brainbarrier which resists the entry of macro-molecules into the subarachnoidspace. Thus, the therapeutic compositions of this invention can beadministered directly to the tumor site. For instance, brain tumors(e.g., gliomas) can be treated by administering the therapeuticcomposition directly to the tumor site (e.g., through a surgicallyimplanted catheter). Where the fluid delivery through the catheter ispressurized, small molecules ( e.g. the therapeutic molecules of thisinvention) will typically infiltrate as much as two to three centimetersbeyond the tumor margin.

[0088] Alternatively, the therapeutic composition can be placed at thetarget site in a slow release formulation (e.g., a thrombin-fibrinogenmixture). Such formulations can include, for example, a biocompatiblesponge or other inert or resorbable matrix material impregnated with thetherapeutic composition, slow dissolving time release capsules ormicrocapsules, and the like.

[0089] Typically the catheter, or catheters, or time release formulationwill be placed at the tumor site as part of a surgical procedure. Thus,for example, where major tumor mass is surgically debulked, theperfusing catheter or time release formulation can be emplaced at thetumor site as an adjunct therapy. Of course, surgical removal of thetumor mass may be undesired, not required, or impossible, in which case,the delivery of the therapeutic compositions of this invention maycomprise the primary therapeutic modality.

[0090] Imaging

[0091] The invention also provides a method of imaging a cell expressinga receptor that binds an hIL13 mutant in vivo. In an exemplary method,an hIL13 mutant conjugated to a label detectable by the chosen imagingtechnique is administered to an animal having the cell expressing areceptor that binds the particular hIL13 mutant. The animal is thenimaged using the chosen imaging technique. Examples of labels useful fordiagnostic imaging include radiolabels such as ¹³¹I, ¹¹¹In, 123I, ⁹⁹mTc,³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh; fluorescent labels such as fluoresceinand rhodamine; nuclear magnetic resonance active labels; positronemitting isotopes detectable by a positron emission tomography (“PET”)scanner; chemiluminescent labels such as luciferin; and enzymaticmarkers such as peroxidase or phosphatase. Mutants of hIL13 can belabeled with such reagents as described above or using techniques knownin the art.

[0092] Any imaging technique compatible with the labeled-hIL13 mutantcan be used. Examples of such techniques include immunoscintigraphywhere a gamma camera is used to detect the location and distribution ofgamma-emitting radioisotopes; MRI where a paramagnetic labeled-hIL13mutant is used; PET where an hIL13 mutant is conjugated with a positronemitting label; and X-ray imaging where an hIL13 mutant is conjugatedwith a radioopaque label (e.g., a metal particle). A more detaileddescription of such techniques is provided in Handbook of TargetedDelivery of Imaging Agents (Handbook of Pharmacology and Toxicology),ed. V. Torchilin, CRC Press, 1995; Armstrong et al., Diagnostic Imaging,Blackwell Science Inc., 1998; and Diagnostic Nuclear Medicine, ed. C.Schiepers, Springer Verlag, 2000.

[0093] As an illustrative example, the location of glioma tumor cells inan animal can be determined by injecting (e.g., parenterally or in situ)an animal with a composition including native hIL13 or an hIL13 mutantconjugated to a detectable label (e.g., a gamma emitting radioisotope).The composition is then allowed to equilibrate in the animal, and tobind to the glioma cells. The animal is then subjected to imaging (e.g.,using a gamma camera) to image where the glioma cells are.

[0094] Diagnostic Kits

[0095] In another embodiment, this invention provides for kits for thetreatment of tumors or for the detection of cells overexpressing IL 13receptors. Kits will typically comprise a chimeric molecule of thepresent invention (e.g., a mutant hIL13 conjugated to a detectablelabel, a mutant hIL13 conjugated to cytotoxin, a mutant IL13 conjugatedto a targeting ligand, etc.). In addition the kits will typicallyinclude instructional materials disclosing means of use of chimericmolecule ( e.g., as a cytotoxin, for detection of tumor cells, toaugment an immune response, etc.). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, where a kit contains a chimeric molecule inwhich the effector molecule is a detectable label, the kit mayadditionally contain means of detecting the label (e.g. enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a sheep anti-mouse-HRP, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

EXAMPLES

[0096] The present invention is further illustrated by the followingspecific examples. The examples are provided for illustration only andare not to be construed as limiting the scope or content of theinvention in any way.

Example 1 Materials and Methods

[0097] Restriction endonucleases and DNA ligase were obtained from NewEngland Biolabs (Beverly, Mass.), Bethesda Research Laboratories (BRL,Gaithersburg, Md.) and Boehringer Mannheim (Indianapolis, Ind.). U.S.E.mutagenesis kit, fast protein liquid chromatographic (FPLC) system,columns and media were obtained from Pharmacia (Piscataway, N.J.).Oligonucleotide primers were synthesized at the Macromolecular CoreLaboratory, Penn State College of Medicine. Polymerase chain reaction(PCR) kit was from Perkin-Elmer Cetus (Norwalk, Conn.). Tissue cultureware was from Corning (Corning, N.Y.).3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(inner salt)/phenazine methasulfate (MTS/PMS) non-radioactive cellproliferation assay was purchased from Promega (Madison, Wis.). SDS-PAGEsupplies were from BioRad (Hercules, Calif.). Antibodies were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, Calif.). SuperSignalSubstrate for chemiluminescent detection was purchased from Pierce(Rockford, Ill.). Cell lines were obtained from the American TypeCulture Collection (Rockville, Md.). MTS/PMS for cell titer 96 aqueousnon-radioactive cell proliferation assay was purchased from Promega(Madison, Wis.).

[0098] For recombinant protein expression in a prokaryotic system, allplasmids carrying the genes encoding proteins of interest were under aT7 promoter-based expression system. The plasmids were constructed asdescribed in Debinski et al., (1998) Nature Biotech., 16:449-453.BL21(1DE3) E. coli, which carry the T7 RNA polymerase gene in anisopropyl-1-thio-b-galactopyranoside (IPTG) inducible form, were used asthe host for recombinant protein expression. Production of recombinantproteins driven by T7 RNA polymerase allowed production of milligramquantities of recombinant protein from a 1.0 liter culture induced atA₆₀₀ of 2.0.

[0099] For expression of proteins, competent BL21 cells were transformedwith the appropriate plasmids and grown in Terrific Broth (DIFCOLaboratories, Detroit Mich.) to A₆₀₀ equal to 2.0, at which point IPTGwas added to a final concentration of 250 mM. Cells were harvested 90min. later. The inclusion body fraction of the cells was isolated anddenatured in 7M guanidine HCl, then renatured by rapid dilution intobuffer, using the disulfide-shuffling method as was previously describedin Debinski et al. (1993) J. Biol. Chem., 268:14065-14070. Afterdialysis, the renatured proteins were purified using a Pharmacia fastprotein liquid chromatography (FPLC) system.

[0100] For mutagenesis, mutations of the hIL13 gene were made bystandard PCR protocols (using the mutated oligonucleotides as sense oranti-sense primers in PCR) or by using a unique site elimination(U.S.E.) mutagenesis kit, based on the procedure developed by Deng andNickoloff in Anal. Biochem., 200:81-88, 1992. Examples of primers usedfor the mutagenesis are shown below in table 1. All mutated plasmidswere isolated and sequenced to verify the correct mutation prior to use.TABLE 1 hIL13.E13D:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGGACCTCATTGAGGAGhIL13.E13I:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGATCCTCATTGAGGAGhIL13.E13K:AGGAGATATACATATGTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGAAGCTCATTGAGGAhIL13.E13R:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGCGCCTCATTGAGGAGhIL13.E13S:TTTGTGTGTCATATGTCCCGAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGTCTCTCATTGAGGAGWP070776;1 hIL13.E13Y:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGTACCTCATTGAGGAGhIL13.E16K:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGGAGCTCATTAAGGAGCTGGThIL13.E17K:TTTGTGTGTCATATGTCCCCAGGCCCTGTGCCTCCTCTACAGCCCTCAGGGAGCTCATTGAGAAGCTGGTCAhIL13.R66D: ATGGAGAAGACCCAGGACATGCTGAGCGGATTC hIL13.D69D:ACCCAGAGGATGCTGGACGGATTCTGCCCGCAC

[0101] For polyacrylamide gel electrophoresis and immunoblotting, thepurity of the isolated recombinant proteins was determined by sodiumdodecyl sulfate polyacrylamide gel electrophoresis, under nonreducingconditions. The separated proteins in the gel were stained either withCoomassie Blue for visual inspection or transferred to polyvinylidenedifluoride (PVDF) membrane for Western blot analysis. For Western blotanalysis, the PVDF with the transferred proteins was incubated in 5%nonfat milk in phosphate buffered saline (PBS) for one hour at roomtemperature. The membrane was incubated for one hour in 5% milk/PBScontaining goat anti-human IL13 antibody (1:1,000 dilution). Theantibody was raised against a hIL13 specific peptide located at thecarboxy terminus of hIL13. After incubation with the primary antibody,the membrane was washed three times, five min. each, with 0.05% Tween20/PBS. The membrane was then incubated for one hour in 5% milk/PBScontaining donkey anti-goat IgG conjugated with horseradish peroxidase(1:20,000 dilution). The membrane was washed three times, five min.each, with 0.05% Tween 20/PBS. The immuno-reactive proteins wereidentified on film, using enhanced chemiluminescence detection. Imageswere digitized using a Hewlett Packard Scan-Jet 6100C scanner andcomposited using Microsoft Powerpoint software.

[0102] For circular dichroism (CD), CD spectra for the proteins wereobtained over the wavelength range of 185-260 nm using a Jasco J-710spectropolarimeter. All measurements were carried at 37° C., using thesame cuvette, the same orientation of the cuvette to the light source,and a 2 mm light path. Proteins (0.1 mg/ml) were resuspended inphosphate buffered saline (PBS) and then analyzed. For unfolded samples,protein was resuspended in 8M urea containing 40 mM DTT (denaturationbuffer). Reported spectra were the average of three consecutive runs foreach sample. Spectra from appropriate blanks, PBS alone or denaturationbuffer, were subtracted from each sample so that the resulting spectrareflected only the CD contribution of the proteins.

[0103] For cell proliferation assays, cell killing by cytotoxins wastested as follows. 5×10³ cells per well were plated in a 96-well tissueculture plate in 150 ml of media. Various concentrations of cytotoxinswere diluted in 0.1% BSA/PBS and 25 ml of each dilution was added tocells 18-24 h following cell plating. Cells were incubated at 37 ° C.for another 48 h. Then, the cytotoxicity was determined using acolorimetric MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt]/PMS (phenazine methasulfate) cell proliferation assay.MTS/PMS was added at a half final concentration as recommended by themanufacturer. The cells were incubated with the dye for 4 hr and thenthe absorbance was measured at 490 nm for each well using a micro-platereader (Cambridge Technology, Inc., Watertown, Mass.). The wellscontaining cells treated with cycloheximide (10 mM) or wells with noviable cells left served as a background for the assay. For blockingstudies, interleukins at a concetration of 1.0 ug/ml were added to cellsfor 60 min before the cytotoxins addition.

[0104] Cell proliferation studies using TF-1 cells (pre-leukemic human Bcells, which express the shared IL13/4 receptor) were performed bygrowing the cells in the presence of different concentrations ofwild-type interleukins or their mutants in 96 well culture plates. After72 h of incubation at 37° C., the rate of proliferation of the TF-1cells was determined by a colorimetric MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt]/PMS (phenazine methasulfate) cell proliferation assay. Thecell samples were incubated with the dye for four h then theirabsorbance at 490 nm was recorded for each well using a microplatereader. The wells with cells treated with high concentrations ofcycloheximide served as background for the assay.

[0105] For indirect immunofluorescence analyses, HUVEC were seeded ontoan eight chambered slide, 50,000 cells per chamber, and incubatedovernight at 37° C. to allow cells to attach. The media was removed andreplaced with media containing hIL13 or its mutants (1 mg/ml finalconcentration). The cells were incubated again overnight at 37° C. Thenext day, the media was removed, the cells were fixed in ethanol andincubated with blocking media (10% normal rabbit serum in PBS) at roomtemperature for 20 min. The blocking media was removed and goatanti-VCAM-1 antibody (1 ug/ml) in 1.5% normal rabbit serum/PBS wasadded. Cells were incubated at room temperature for one hour, thenprimary antibody was removed and cells rinsed three times, five min.each, with PBS. Cells were incubated with rabbit anti-goat IgG-Cy3conjugate (1:150 dilution) in 1.5% normal rabbit serum/PBS for 45 min.at room temperature, in the dark. After 45 min., the cells were rinsedthree times, five min. each, with PBS, a coverslip was mounted usingaqueous mounting medium, and the fluorescent staining determined using arhodamine filter set. Images were obtained from the same experimentwithout adjusting the microscope between samples on a Zeiss Axioplanmicroscope and captured digitally using Snappy by Play Inc.

[0106] For cytotoxicity-blocking assays, glioblastoma cells (U-251 MGand SNB-19) were plated into 96-well culture plates and incubated for 24h. After 24 h, hIL13 or its mutants were added to cells and incubatedfor one hour at 37° C. An equal volume of 0.1% BSA in PBS was added tocells for assays without blocking ligand. After the hour incubation,increasing concentration of the hIL13 chimeric toxin (hIL13-PE1E; seeDebinski, et al. (1996) J. Biol. Chem., 271, 22428-22433)) was added(0.001-10 ng/ml final concentration) and the cells were incubated forthree days. After three days, the number of proliferating cells in eachwell was determined using the calorimetric MTS/PMS method describedabove. The wells with cells treated with high concentrations ofcycloheximide served as background for the assay.

[0107] For autoradiography, recombinant hIL13.E13Y was iodo-labeled with¹²⁵I by using the IODO-GEN reagent (Pierce) according to themanufacturer's instructions. The specific activity of ¹²⁵I-hIL13.E13Ywas ˜300 mCi/mg of protein. All studies involving human specimens wereapproved by the respective Human Subjects Protection Offices at the PennState College of Medicine (Protocol No. IRB 96-123EP). Serial tissuesections were cut (10 mm) on a cryostat, thaw-mounchrome alumme-alumcoated slides, and stored at 4° C. until analyzed. To observe bindingdistribution of ¹²⁵I-hIL13.E13Y, sections were incubated (1 hr, 22° C.)with 1.0 nM ¹²⁵I-hIL13 in binding buffer (200 mM sucrose, 50 mM HEPES,1% BSA, 10 mM EDTA). Adjacent serial sections were incubated with theradiolabeled recombinant hIL13.E13Y after a 30 min pre-incubation at 22°C. in the presence of binding buffer alone or of a 100- to 500-foldmolar excess of unlabeled hIL13, hIL13.E13Y or hIL4, or a monoclonalantibody against human transferrin receptor (TfR). To dissociatenon-specifically bound radioligand, sections were rinsed in fourconsecutive changes (5 minutes each) of ice-cold 0.1 M PBS. At least twosections of each of the tissue specimens were assayed for the evaluationof ¹²⁵I-hIL13.E13Y binding specificity. After drying, labeled sectionswere apposed to Kodak autoradiography film at −65° C. for 8 hr to 11days.

Example 2 Radioimmunodetection and Radioimmunotherapy of Human HighGrade Gliomas

[0108] The IL13 mutein, hIL13.E13Y, was prepared as described above andtested for its ability to modulate the interleukin-induced proliferativeresponses of TF-1 cells. TF-1 cells were treated with hIL13, hIL13.E13Y,or hIL13.E13K. While hIL13 was very potent in stimulating the growth ofTF-1 cells, hIL13.E13K showed no activity and hIL13.E13Y exhibited onlyvery weak activity, if any at all.

[0109] The ability of hIL13.E13Y to compete for the hIL13 binding sitesin clinical specimens of glioblastoma (GBM) in situ was investigated inautoradiographic studies. The two GBM tissues studied labeled denselywith ¹²⁵I-hIL13.E13Y binding sites, as well as with labeled wild typehIL13. The binding was specific since both hIL13.E13Y and the wild typeIL13 blocked the binding of ¹²⁵I-hIL13.E13Y. In contrast, an excess ofrecombinant hIL4 was largely without influence on the ¹²⁵I-hIL13.E13Ybinding to GBM specimens.

[0110] In another test of specificity of the hIL13.E13Y binding to GBM,the ability of a monoclonal antibody against the transferrin receptor(TfR) to displace the binding of radiolabeled interleukin was examined.No cross-competition for the hIL13 binding sites in the GBMs examinedwas observed. The binding of hIL13.E13Y to GBM appears to be veryspecific as others studies have shown that ¹²⁵-hIL13 fails to interactwith normal brain or normal human cells and that ¹²⁵I-hIL13.E13Y doesnot interact with normal human cells, such as HUVEC.

[0111] In other tests, the ability of hIL13.E13Y to block the action ofhIL13-PE1E (an hIL13-based cytotoxin) was investigated using twodifferent human malignant glioma cell lines. Glioma cells in culturewere pretreated with either hIL13, hIL13.E13Y or hIL13.E13K beforehIL13-PE1E was added. The cytotoxicity of hIL13-PE1E was neutralized inthese cultures using hIL13, hIL13.E13Y, or hIL13.E13K.

Example 3 Mutants of Interleukin 13 with Altered Reactivity TowardsInterleukin 13 Receptors

[0112] Recombinant IL-13 and IL-13 mutants were prepared, isolated, andpurified as described in Example 1. The prokaryotic production of thecytokines or their mutants under control of the T7 promoter was veryefficient. After purification, between 0.5 mg and 1.5 mg of eachcytokine or mutant was obtained from a 1 liter culture. When eachpurified protein was analyzed using SDS-PAGE and stained with CoomassieBlue, a single protein band was observed migrating at approximately 13kDa (FIG. 1, panel A). Visual inspection suggested that all preparationswere greater than 95% pure. A corresponding Western blot of the samplesusing a goat polyclonal anti-hIL13 antibody (that was notcross-reactivity with any other cytokine) indicated that the isolatedproteins were immuno-reactive with hIL13 (FIG. 1, panel B). Thealpha-helix D mutants, hIL13.R109D and hIL13.F113D, also reacted withthis antibody indicating that they too were immuno-reactive with hIL13(data not shown). Traces of a dimeric form (˜26 kDa) of some of themutated cytokines were also detected.

[0113] To determine whether the recombinant interleukins had refoldedcorrectly and that their mutation had not destroyed their generalpattern of conformation, circular dichroism (CD) was used to determinethe proteins'folded structure. The secondary structure data from thespectropolarimeter indicated that each protein sample produced aspectrum consistent with an alpha-helical enriched protein, having twospectral minima at approximately 208 nm and 222 nm (FIG. 2).Furthermore, the CD spectrum of each mutant could be super-imposed onthe CD spectrum of the wild-type hIL13, although slight variations inspectra intensity were observed between samples (FIG. 2, panels A, B,C). hIL13.R109D and hIL13.F113D both produced CD spectra similar to theother mutants (not shown). For comparison, the CD spectrum of unfoldedhIL13 was also obtained (FIG. 2, panel D). The panel illustrates thecollapse of the characteristic alpha-helical pattern when the protein isunfolded.

[0114] Functional assays were employed to examine whether the IL13mutants exhibited an altered association with the shared signalingIL13/4 receptor by measuring their effect on induced TF-1 cellproliferation. TF-1 cells express the shared IL13/4 receptor (but notthe restricted receptor) and proliferate in a dose-dependent manner inthe presence of hIL13 or hIL4. Under the conditions used in this assay,a concentration of 100 ng/ml of wild-type hIL13 consistently produced amaximal proliferative response in TF-1 cells of ˜300% that of thebaseline value (FIG. 3, panel A). Differences were observed in TF-1 cellproliferation depending on whether the mutants were in the predictedalpha-helices A, C, or D. Of the alpha-helix A mutants, hIL13.E13Kinduced only a minimal proliferative response over the range tested(FIG. 3, panel B), and hIL13.E13I, hIL13.E13S, and hIL13.E13Y failed toinduce any proliferative response (FIG. 3, panel B). Mutants hIL13.E13Dand, unexpectedly, hIL13.E13R both induced a dose-dependent dependentincrease in proliferation of the TF-1 cells. Their induction of TF-1cell proliferation followed the same pattern as wild-type hIL13,although hIL13.E13D had a lesser effect on proliferation than didhIL13.E13R (FIG. 3, panel A). Both hIL13.E16K and hIL13.E17K (withmutated sites one turn of the alpha-helix up from position 13) induced adose-dependent increase in the proliferative response of the TF-1 cells(FIG. 3, panel A). While the hIL13.E17K-induced effect was comparable towild-type hIL13, the hIL13.E16K-induced effect was significantly greaterthan that caused by wild-type hIL13.

[0115] The alpha-helix C mutants, hIL13.R66D and hIL13.S69D, both showeda significantly impaired ability to stimulate TF-1 cells, compared towild-type hIL13 (FIG. 3, panel C) Their action on TF-1 cells, however,can be classified between that caused by mutants shown in FIG. 3, panelsA and B. The alpha-helix D mutants also exhibited contrasting patternsof action on TF-1 cells. The hIL13.F113D mutant was equivalent towild-type hIL13 in inducing TF-1 cell proliferation, while thehIL13.R109D mutant was inactive on these cells (not shown).

[0116] The ability of the hIL13 mutants to interact with the sharedhIL13/4 receptor on normal cells was assessed by examining their effecton VCAM-1 expression on the surface of HUVEC. Cytokine binding of theshared IL13/4 receptor on the HUVEC cell surface results intransmembrane signaling events that induce VCAM-1 expression on thesecells. Results from two separate experiments are shown in FIG. 4. Cellsincubated in the absence of hIL13 showed minimal, nonspecific VCAM-1staining (FIG. 4, panels A and G). In contrast, cells incubatedovernight in media containing wild-type hIL13 exhibited a markedincrease in VCAM-1 (FIG. 4, panels B and H). The pattern of the stainingappeared to be specific for certain areas of the cell surface, comparedto the minimal, homogeneous staining of cells that had not beenincubated with cytokine (FIG. 4, panels A and G). Cells incubated withmutants hIL13.E13I, hIL13.E13K, and hIL13.E13Y, which are unable toinduce TF-1 cell proliferation (FIG. 3), showed less VCAM-1 expressionthan those treated with wild-type hIL13 (FIG. 4, panels C, D, F, and B,respectively). Although mutant hIL13.F113D was not tested, thehIL13.R109D-induced VCAM-1 staining was negligible (not shown),suggesting again the involvement of alpha-helix D of the cytokine ineffective signaling through the shared receptor. Cells treated withmutants hIL13.E13R and hIL13.E17K showed an increase in VCAM-1 stainingsimilar to that induced by wild-type hIL13, when compared to theirrespective controls (FIG. 4, panels E and J). Mutant hIL13.E16K appearedto have a superagonistic effect on VCAM-1 expression compared to itswild-type IL13 control (FIG. 4, panels I and H, respectively).

[0117] The ability of hIL13 and its mutants to block thecancer-restrictive hIL13 receptor on two different human glioblastomacell lines was examined in cytotoxicity assays using hIL13-PE1E, anextremely potent anti-tumor agent on glioma cells (see Debinski et al.(1996) J. Biol. Chem., 271: 22428-22433). The cytotoxin caused a highlevel of cytotoxicty in cultured U-251-MG cells (FIG. 5, panel A) andSNB-19 cells (FIG. 5, panel B) when the cells were cultured in theabsence of a competing ligand for the receptor. When cultured in thepresence of hIL13 or any of its A or C helix mutants, the level ofcytotoxicity was reduced even at the highest concentration of cytotoxinused (FIG. 5, panels A and B). IC₅₀s for tests without blocking ligandwere 0.1 ng/ml (1.25 pM) for U-251-MG cells and 0.07 ng/ml (0.875 pM)for SNB-19 cells. In contrast, the blocking assay using hIL13 mutantsshowed their ability to increase the IC₅₀ by at least 100 times. Forconcentrations of hIL13 or its mutants up to 1000×(by weight) overhIL13-PE1E, no discernable differences were detected between thesevarious mutants and wild-type hIL13 in blocking the cytotoxin's activityon the glioma cells (FIG. 5, panels A and B). hIL13.F113D, analpha-helix D mutant, behaved as the wild-type cytokine. In contrast,addition of hIL13.R109D to the cell cultures did not reduce thecytotoxin-induced cytotoxicity. hIL4 did not display any neutralizingactivity in these assays.

[0118] Other Embodiments

[0119] This description has been by way of example of how thecompositions and methods of invention can be made and carried out. Thoseof ordinary skill in the art will recognize that various details may bemodified in arriving at the other detailed embodiments, and that many ofthese embodiments will come within the scope of the invention.

[0120] Therefore, to apprise the public of the scope of the inventionand the embodiments covered by the invention, the following claims aremade.

1 23 1 114 PRT Homo sapiens 1 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 2 114 PRT ARTIFICIALSEQUENCE misc_feature hIL13 mutant having a Glu to Lys substitution atresidue 13 2 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Lys Leu IleGlu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu CysAsn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr CysAla Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile GluLys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser AlaGly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu ValAla Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe ArgGlu Gly Arg 100 105 110 Phe Asn 3 114 PRT ARTIFICIAL SEQUENCEmisc_feature hIL13 mutant having a Glu to Ile substitution at residue 133 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Ile Leu Ile Glu 1 5 1015 Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 20 2530 Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 35 4045 Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 5560 Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln 65 7075 80 Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 8590 95 Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg100 105 110 Phe Asn 4 114 PRT ARTIFICIAL SEQUENCE misc_feature hIL13mutant having a Glu to Cys substitution at residue 13 4 Ser Pro Gly ProVal Pro Pro Ser Thr Ala Leu Arg Cys Leu Ile Glu 1 5 10 15 Glu Leu ValAsn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met ValTrp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu SerLeu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg MetLeu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln 65 70 75 80 Phe SerSer Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val LysAsp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg 100 105 110 PheAsn 5 114 PRT ARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Gluto Ser substitution at residue 13 5 Ser Pro Gly Pro Val Pro Pro Ser ThrAla Leu Arg Ser Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln AsnGln Lys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn LeuThr Ala Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val SerGly Cys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe CysPro His Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val ArgAsp Thr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu HisLeu Lys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 6 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Glu to Argsubstitution at residue 13 6 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala LeuArg Arg Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn Gln LysAla Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu Thr AlaGly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser Gly CysSer Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys Pro HisLys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg Asp ThrLys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His Leu LysLys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 7 114 PRT ARTIFICIALSEQUENCE misc_feature hIL13 mutant having a Glu to Tyr substitution atresidue 13 7 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Tyr Leu IleGlu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu CysAsn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr CysAla Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile GluLys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser AlaGly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu ValAla Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe ArgGlu Gly Arg 100 105 110 Phe Asn 8 114 PRT ARTIFICIAL SEQUENCEmisc_feature hIL13 mutant having a Glu to Asp substitution at residue 138 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Asp Leu Ile Glu 1 5 1015 Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 20 2530 Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 35 4045 Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 5560 Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln 65 7075 80 Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 8590 95 Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg100 105 110 Phe Asn 9 114 PRT ARTIFICIAL SEQUENCE misc_feature hIL13mutant having a Glu to Lys substitution at residue 16 9 Ser Pro Gly ProVal Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile Lys 1 5 10 15 Glu Leu ValAsn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met ValTrp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu SerLeu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg MetLeu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln 65 70 75 80 Phe SerSer Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val LysAsp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg 100 105 110 PheAsn 10 114 PRT ARTIFICIAL SEQUENCE misc_feature hIL13 mutant having aGlu to Lys substitution at residue 17 10 Ser Pro Gly Pro Val Pro Pro SerThr Ala Leu Arg Glu Leu Ile Glu 1 5 10 15 Lys Leu Val Asn Ile Thr GlnAsn Gln Lys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile AsnLeu Thr Ala Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn ValSer Gly Cys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly PheCys Pro His Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His ValArg Asp Thr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu LeuHis Leu Lys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 11 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Arg to Aspsubstitution at residue 66 11 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Asp Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 12 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Ser to Aspsubstitution at residue 69 12 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Asp Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 13 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Asp to Lyssubstitution at residue 99 13 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Lys Leu Leu Leu His LeuLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 14 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Leu to Alasubstitution at residue 102 14 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Ala His LeuLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 15 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Leu to Alasubstitution at residue 104 15 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His AlaLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 16 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Lys to Aspsubstitution at residue 105 16 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuAsp Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 17 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Lys to Aspsubstitution at residue 106 17 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Asp Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 18 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Leu to Alasubstitution at residue 107 18 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Ala Phe Arg Glu Gly Arg 100 105 110 Phe Asn 19 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Phe to Tyrsubstitution at residue 108 19 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Tyr Arg Glu Gly Arg 100 105 110 Phe Asn 20 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Arg to Aspsubstitution at residue 109 20 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Phe Asp Glu Gly Arg 100 105 110 Phe Asn 21 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Arg to Aspsubstitution at residue 112 21 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Phe Arg Glu Gly Asp 100 105 110 Phe Asn 22 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having a Phe to Aspsubstitution at residue 113 22 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Asp Asn 23 114 PRTARTIFICIAL SEQUENCE misc_feature hIL13 mutant having an Asn to Aspsubstitution at residue 113 23 Ser Pro Gly Pro Val Pro Pro Ser Thr AlaLeu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn GlnLys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu ThrAla Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser GlyCys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys ProHis Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg AspThr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His LeuLys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asp

What is claimed is:
 1. An antibody that specifically binds a mutanthIL13 molecule but not a native hIL13 molecule.
 2. The antibody of claim1, wherein the antibody is a monoclonal antibody.
 3. The antibody ofclaim 1, wherein the antibody is a polyclonal antibody.
 4. The antibodyof claim 1, wherein the mutant hIL13 is a polypeptide consisting of anamino acid sequence selected from the group consisting of SEQ ID Nos:2-23.
 5. The antibody of claim 4, wherein the mutant hIL13 is apolypeptide consisting of the amino acid sequence of SEQ ID NO:
 2. 6.The antibody of claim 4, wherein the mutant hIL13 is a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 3. 7. The antibodyof claim 4, wherein the mutant hIL13 is a polypeptide consisting of theamino acid sequence of SEQ ID NO:
 4. 8. The antibody of claim 4, whereinthe mutant hIL13 is a polypeptide consisting of the amino acid sequenceof SEQ ID NO:
 5. 9. The antibody of claim 4, wherein the mutant hIL13 isa polypeptide consisting of the amino acid sequence of SEQ ID NO:
 6. 10.The antibody of claim 4, wherein the mutant hIL13 is a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 7. 11. The antibodyof claim 4, wherein the mutant hIL13 is a polypeptide consisting of theamino acid sequence of SEQ ID NO:
 8. 12. The antibody of claim 4,wherein the mutant hIL13 is a polypeptide consisting of the amino acidsequence of SEQ ID NO:
 9. 13. The antibody of claim 4, wherein themutant hIL13 is a polypeptide consisting of the amino acid sequence ofSEQ ID NO:
 10. 14. The antibody of claim 4, wherein the mutant hIL13 isa polypeptide consisting of the amino acid sequence of SEQ ID NO: 11.15. The antibody of claim 4, wherein the mutant hIL13 is a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 12. 16. The antibodyof claim 4, wherein the mutant hIL13 is a polypeptide consisting of theamino acid sequence of SEQ ID NO:
 13. 17. The antibody of claim 4,wherein the mutant hIL13 is a polypeptide consisting of the amino acidsequence of SEQ ID NO:
 14. 18. The antibody of claim 4, wherein themutant hIL13 is a polypeptide consisting of the amino acid sequence ofSEQ ID NO:
 15. 19. The antibody of claim 4, wherein the mutant hIL13 isa polypeptide consisting of the amino acid sequence of SEQ ID NO: 16.20. The antibody of claim 4, wherein the mutant hIL13 is a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 17. 21. The antibodyof claim 4, wherein the mutant hIL13 is a polypeptide consisting of theamino acid sequence of SEQ ID NO:
 18. 22. The antibody of claim 4,wherein the mutant hIL13 is a polypeptide consisting of the amino acidsequence of SEQ ID NO:
 19. 23. The antibody of claim 4, wherein themutant hIL13 is a polypeptide consisting of the amino acid sequence ofSEQ ID NO:
 20. 24. The antibody of claim 4, wherein the mutant hIL13 isa polypeptide consisting of the amino acid sequence of SEQ ID NO: 21.25. The antibody of claim 4, wherein the mutant hIL13 is a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 22. 26. The antibodyof claim 4, wherein the mutant hIL13 is a polypeptide consisting of theamino acid sequence of SEQ ID NO: 23.